1. Field of the Invention
The present invention relates generally to the fields of biochemical endocrinology, molecular biology and protein chemistry. More specifically, the present invention relates to novel methods for the characterization of compounds which stimulate STF-1 expression in pancreatic islet cells.
2. Description of the Related Art
Glucose homeostasis requires the concerted efforts of numerous neuroendocrine systems. The pancreatic islets are considered to be the primary "glucose sensor" in mammals. Pancreatic islets contain four populations of cells which are characterized primarily as insulin, glucagon, somatostatin or pancreatic polypeptide-producing cells. Among these, insulin-producing .beta.-cells predominate. Insulin secretion and production are stimulated by increases in serum glucose, an event which i s mandatory for subsequent glucose uptake in certain tissues. Hence, dysfunction or destruction of .beta.-cells results in elevated serum glucose levels, ultimately developing into diabetes mellitus.
Genetic linkage analysis indicates that hereditary factors strongly influence susceptibility to acquisition of the diabetic state. For example, at least 18 genetic loci have some degree of linkage to insulin-dependent diabetes mellitus (IDDM). One disease susceptibility locus, termed IDDM2, encompasses the human insulin gene and is associated with altered transcriptional regulation of insulin promoter function. Hence, disruption of the processes that regulate insulin gene expression may account in part for diabetogenesis. Consistent with this hypothesis, impaired .beta.-cell function is a very common feature of diabetes.
Non-insulin dependent diabetes mellitus (NIDDM) i s thought to occur as a result of both external and complex genetic influences. Interestingly, allelic variants at the insulin locus itself have been associated with the disease. These variants appear to contain a normal insulin gene, but exhibit altered properties with regard to transcriptional regulation.
Estimates indicate that as many as 20 million Americans may suffer from non-insulin dependent diabetes mellitus (Type II) diabetes. The progression of the disease appears to require both environmental factors and certain as yet largely unidentified diabetes susceptibility genes, which may contribute to the peripheral insulin resistance of Type II diabetes, in which tissues fail to utilize glucose appropriately in response to the insulin signal. Alternatively, genetic factors may account for the reduced glucose sensitivity of the insulin-producing pancreatic .beta.-cells in these individuals. The end result of both of these physiological states is the marked hyperglycemia which constitutes the primary hallmark of diabetes.
Transcriptional control of the insulin gene is achieved through a short region of flanking DNA that interacts with cell-specific and glucose-sensitive signaling molecules. The precise nature of this regulatory organization remains poorly understood, although it is generally acknowledged that basic helix-loop-helix (bHLH) and homeodomain-containing factors are critical components of the transcriptional machinery that governs .beta.-cell-specific expression of insulin. An islet-specific basic helix-loop-helix complex interacts with a proximal E-box that has been variously termed Nir, IEBI or ICE; this element is present twice in the rat insulin I gene, but only once in the rat insulin II and human insulin genes.
Transient assays in insulin-producing cell lines suggest that E-box-binding factors synergize with .beta.-cell-specific proteins that bind a nearby AT-rich sequence termed FLAT, which bears the hallmarks of a homeodomain recognition sequence. Several characterized homeodomain proteins have been shown to bind the FLAT element, including Isl-1, 1mx-1, cdx-3 and STF-1. In addition, the latter of these corresponds to the principal binding activity at a n evolutionarily conserved AT-rich sequence termed the P-element. Isl-1 binds the FLAT element weakly and does not appear to be present in the FLAT-binding complexes detected with extracts from insulin-producing cells. Current evidence supports a more important role for Isl-1 in neural development. The homeodomain factors 1mx-1 and cdx-3 have interesting transactivation properties with regard to insulin promoter function in heterologous cells, but their cellular distribution and FLAT-binding ability inside the .beta.-cell remains unclear. In addition, there is little data that directly address the function of these factors in .beta.-cell lines.
Within the group of factors with insulin promoter-binding activity, STF-1 is perhaps the most promising candidate for a bona fide regulator of insulin promoter function. In mice, STF-1 is first detected at embryonic day 8.5 in the nuclei of primordial cells that gives rise to the pancreas, shortly prior to the earliest detected expression of insulin in this region. Throughout the ensuing development of the endocrine pancreas, STF-1 and insulin are largely co-expressed. In addition, in extracts from insulin-producing cells lines, STF-1 appears to be a component of the endogenous DNA-binding activity at both the FLAT and P elements in the insulin promoter. STF-1 also strongly synergizes with the E-box-binding factor Pan-1, as might be expected from a FLAT-binding factor. However, DNA-binding assays indicate that other, unknown, factors from .beta.-cell extracts also make a large contribution to the detected FLAT-binding activity. It remains unclear whether FLAT-mediated insulin promoting activity requires all, or only a subset, of these detected species.
Although STF-1 is initially expressed in both exocrine and endocrine cells of the developing pancreas, the production of STF-1 is progressively restricted to insulin- and somatostatin-producing islet cells. In these cells, STF-1 action appears to be important for maintaining high level expression of both somatostatin and insulin genes.
STF-1 recognizes two well defined islet-specific elements on the insulin promoter, termed FLAT and P. When bound to these sites, STF-1 stimulates insulin transcription in concert with E47, a helix-loop-helix protein which recognizes two E-box elements termed Far and Nir. Similarly, STF-1 regulates somatostatin expression in islet cells via two islet specific elements, termed TSEI and TSEII.
Homeodomain proteins such as STF-1 have been found to play an important role in development by establishing cell or segmental identity. In contrast to their specific and distinct effects in vivo, most homeodomain proteins exhibit low and overlapping DNA binding specificity in vitro. However, recent studies have implicated certain protein co-factors as determinants of homeodomain DNA binding specificity in vivo. In Drosophila, for example, extradenticle (exd) has been shown to modulate the activity of homeotic proteins without altering their pattern of expression. Rather, extradenticle appears to promote target gene selection by enhancing the DNA binding specificity of certain homeodomain proteins. Indeed, extradenticle is highly conserved in vertebrates, sharing extensive sequence similarity (71%) with the human protooncogene Pbx1.
The commitment of cells to specific lineages during development is determined in large part by the relative expression of various homeodomain (HOX) selector proteins which mediate the activation of distinct genetic programs. But the mechanisms by which individual HOX genes are themselves targeted for expression in different cell types remains largely uncharacterized.
The vertebrate pancreas consists of endocrine and exocrine components which arise from a common progenitor cell in the duodenal anlage (1). Within the endocrine component of the pancreas, a pluripotent precursor cell which initially expresses multiple islet hormones undergoes progressive restriction to form the four subpopulations of cells comprising the adult islets of Langerhans: insulin, somatostatin, glucagon, and pancreatic polypeptide-producing cells (2, 3). The mechanism by which these developmental pathways are activated is unclear, but current evidence implicates the homeobox factor STF-1 (IPF-1/IDX-1) as an important determinant in this process. Indeed, the requirement for STF-1 in development is supported by homologous recombination studies in which targeted disruption of the STF-1/IPF-1 gene leads to congenital absence of the pancreas (4).
STF-1 (also referred to as Pdx1) expression is first detectable at embryonic day 8.5 in cells of the pancreatic anlage and in pluripotent precursor cells. Transiently expressed in both endocrine and exocrine components of the developing pancreas, STF1 production is progressively restricted to insulin and somatostatin producing islet cells (5, 6). In these cells, STF-1 appears to regulate both insulin and somatostatin genes by binding to functional elements within each promoter (5, 7-16).
The prior art is deficient in the lack of effective means of regulating expression of the homeodomain protein STF-1 in pancreatic cells. The present invention fulfills this longstanding need and desire in the art.